The long-term goal of this project is to better understand drug-drug interactions that arise primarily from the inhibitory effects of drug metabolites, rather than from the effects of the drugs themselves. It is well established that metabolites of drugs can be toxic, pharmacologically active, a cause of clinically significant drug-drug interactions and/or the source of non-linear drug pharmacokinetics. Thus, metabolites often play an important role in therapeutics. Metabolite-based drug interactions that arise from the inhibitory effects of drug metabolites, rather than the parent drugs, on P450 enzyme activity are poorly understood and difficult to predict. Rationalization of these types of interaction requires simultaneous resolution of two of the major scaling issues that plague drug metabolism research - (1) the application of in vitro data for a drug in order to predict in vivo metabolite pharmacokinetics and (2) prediction of the magnitude of a drug-drug interaction caused by a drug (in this case a metabolite) in vivo. The goal of this application is to establish theoreticallybased and experimentally verified means to understand, detect and more reliably predict metabolicallybased drug interactions. Aim 1 completes our in vivo and in vitro work on the stereoselective metabolism of itraconazole (a potent inhibitor of drug metabolism in vivo) and the effects of the long lived, tight binding metabolites on enzyme activity and dose dependent pharmacokinetics of the parent drug. Aim 2 focuses on a rationalization of the magnitude of drug-drug interactions that occur as the result of long term dosing of fluoxetine with drugs that are cleared by CYP2D6, CYP2C19 and CYP3A4 and the role of fluoxetine metabolites in these interactions. Experimental approaches use single recombinant enzymes, hepatocytes and in vivo interaction studies to test our predictions. Aim 3 extends our work on the complexities of the irreversible inhibition of CYP3A4 by diltiazem and it's metabolites in single enzyme systems, microsomes and hepatocytes. The major objective here is to establish and rationalize new methods to meaningfully quantify in vitro behavior when the inhibition is almost entirely driven by the concentrations of diltiazem's metabolites. .Successful completion of these aims will provide powerful new tools to use in the prospective evaluation of metabolite-based drug interactions.